Etiologic Risk Group (see link below): Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
*Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium avium, subspecies paratuberculosis (MAP) is a bacterial chronic disease of the intestines. In animals, such as diary cows, the disease presents itself as the Johne’s disease. However, MAP has been traced to be the single etiologic agent of the causation of Crohn’s disease and ulcerative colitis in humans. Classified as an IBD (inflammatory bowel diseases), Crohn’s can cause rectal bleeding, persistent diaherra, fever, and loss of appetite. http://www.gutpathogens.com/content/2/1/21
Essentiality of this protein: AckA is essential for catalytic and metabolic activities of e-coli, and renders those two time-dependent without ackA. http://www.ncbi.nlm.nih.gov/pubmed/6252971
Acetate activity will be determined by presence of pyruvate with a continuous spectrophotometric rate determination at 340 nm absorbance wavelength. Reagents include 100 mM Triethanolamine Buffer, 1 M Sodium Acetate Solution (NaOAc), 91 mM Adenosine 5'-Triphosphate Solution (ATP), 56 mM Phospho(enol)pyruvate Solution (PEP), 200 mM Magnesium Chloride Solution (MgCl2), 6.4 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form Solution (ß-NADH), PK/LDH Enzymes Suspension, Myokinase Enzyme Solution (MK), and Acetate Kinase Enzyme Solution. “One unit will phosphorylate 1.0 µmole of acetate to acetyl phosphate per minute at pH 7.6 at 25°C.”
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MDGSDGARRVLVINSGSSSLKFQLVDPEFGVAASTGIVERIGEESSPVPDHDAALRRAFD MLAGDGVDLNTAGLVAVGHRVVHGGNTFYRPTVLDDAVIARLHELSELAPLHNPPALQGI EVARRLLPDIAHVAVFDTGFFHDLPPAAATYAIDRELADRWQIRRYGFHGTSHRYVSEQA AAFLDRPLRGLKQIVLHLGNGCSASAIAGTRPLDTSMGLTPLEGLVMGTRSGDIDPSIVS YLCHTAGMGVDDVESMLNHRSGVVGLSGVRDFRRLRELIESGDGAAQLAYSVFTHRLRKY IGAYLAVLGHTDVISFTAGIGENDAAVRRDAVSGMEELGIVLDERRNLAGGKGARQISAD DSPITVLVVPTNEELAIARDCVRVLGG
**<span style="font-family: Calibri,sans-serif; font-size: 11pt;">*length of your protein in Amino Acids:</span>**<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">386</span>
**<span style="font-family: Calibri,sans-serif; font-size: 11pt;">Molecular Weight of your protein in kiloDaltons using the <span style="color: windowtext; text-decoration: none;">[[http://expasy.org/tools/protparam.html|Expasy ProtParam]]</span> website</span>**<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">41194.6</span>
**<span style="font-family: Calibri,sans-serif; font-size: 11pt;">Molar Extinction coefficient of your protein at 280 nm wavelength:</span>**<span style="color: #333333; font-family: Helvetica,sans-serif; font-size: 10.5pt;">17545</span>
*GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized): Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Acetate Kinase (ackA MAP_3886)
*NCBI Gene # or RefSeq#:
262316 [NCBI]
*Protein ID (NP or XP #) or Wolbachia#:
NP_962820.1.
*Organism (including strain):
Mycobacterium avium subsp. paratuberculosis
Etiologic Risk Group (see link below):
Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
*Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium avium, subspecies paratuberculosis (MAP) is a bacterial chronic disease of the intestines. In animals, such as diary cows, the disease presents itself as the Johne’s disease. However, MAP has been traced to be the single etiologic agent of the causation of Crohn’s disease and ulcerative colitis in humans. Classified as an IBD (inflammatory bowel diseases), Crohn’s can cause rectal bleeding, persistent diaherra, fever, and loss of appetite.
http://www.gutpathogens.com/content/2/1/21
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=5628
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/protein/NP_962820.1?report=genpept
Essentiality of this protein:
AckA is essential for catalytic and metabolic activities of e-coli, and renders those two time-dependent without ackA.
http://www.ncbi.nlm.nih.gov/pubmed/6252971
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):
2 amino acid chains and 4 ligands in ASU
Complex of proteins?:
“2-mer”
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ncbi.nlm.nih.gov/pubmed/23928841
http://www.ncbi.nlm.nih.gov/pubmed/6252971
*EC#:
2.7.2.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.2.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Spectrophotometric
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/acetate_kinase.pdf
Acetate activity will be determined by presence of pyruvate with a continuous spectrophotometric rate determination at 340 nm absorbance wavelength. Reagents include 100 mM Triethanolamine Buffer, 1 M Sodium Acetate Solution (NaOAc), 91 mM Adenosine 5'-Triphosphate Solution (ATP), 56 mM Phospho(enol)pyruvate Solution (PEP), 200 mM Magnesium Chloride Solution (MgCl2), 6.4 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form Solution (ß-NADH), PK/LDH Enzymes Suspension, Myokinase Enzyme Solution (MK), and Acetate Kinase Enzyme Solution. “One unit will phosphorylate 1.0 µmole of acetate to acetyl phosphate per minute at pH 7.6 at 25°C.”
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:3R9P
Current Inhibitors:
trifluoroethanol (TFE), trifluoroethyl butyrate, N-ethylmaleimide
http://www.ncbi.nlm.nih.gov/pubmed/6252971http://www.ncbi.nlm.nih.gov/pubmed/23928841
Expression Information (has it been expressed in bacterial cells):
Expressed in E. coli cells
Purification Method:
Ammonium sulfate fractionation
Gel filtration
Diethylaminoethyl-cellulose chromatography
Affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B
http://www.researchgate.net/publication/22919157_Purification_and_properties_of_acetate_kinase_from_Acholeplasma_laidlawii
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MDGSDGARRVLVINSGSSSLKFQLVDPEFGVAASTGIVERIGEESSPVPDHDAALRRAFD
MLAGDGVDLNTAGLVAVGHRVVHGGNTFYRPTVLDDAVIARLHELSELAPLHNPPALQGI
EVARRLLPDIAHVAVFDTGFFHDLPPAAATYAIDRELADRWQIRRYGFHGTSHRYVSEQA
AAFLDRPLRGLKQIVLHLGNGCSASAIAGTRPLDTSMGLTPLEGLVMGTRSGDIDPSIVS
YLCHTAGMGVDDVESMLNHRSGVVGLSGVRDFRRLRELIESGDGAAQLAYSVFTHRLRKY
IGAYLAVLGHTDVISFTAGIGENDAAVRRDAVSGMEELGIVLDERRNLAGGKGARQISAD
DSPITVLVVPTNEELAIARDCVRVLGG
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atggatggcagcgatggcgcgcgccgcgtgctggtgattaacagcggcagcagcagcctg aaatttcagctggtggatccggaatttggcgtggcggcgagcaccggcattgtggaacgc
attggcgaagaaagcagcccggtgccggatcatgatgcggcgctgcgccgcgcgtttgat atgctggcgggcgatggcgtggatctgaacaccgcgggcctggtggcggtgggccatcgc
gtggtgcatggcggcaacaccttttatcgcccgaccgtgctggatgatgcggtgattgcg cgcctgcatgaactgagcgaactggcgccgctgcataacccgccggcgctgcagggcatt
gaagtggcgcgccgcctgctgccggatattgcgcatgtggcggtgtttgataccggcttt tttcatgatctgccgccggcggcggcgacctatgcgattgatcgcgaactggcggatcgc
tggcagattcgccgctatggctttcatggcaccagccatcgctatgtgagcgaacaggcg gcggcgtttctggatcgcccgctgcgcggcctgaaacagattgtgctgcatctgggcaac
ggctgcagcgcgagcgcgattgcgggcacccgcccgctggataccagcatgggcctgacc ccgctggaaggcctggtgatgggcacccgcagcggcgatattgatccgagcattgtgagc
tatctgtgccataccgcgggcatgggcgtggatgatgtggaaagcatgctgaaccatcgc agcggcgtggtgggcctgagcggcgtgcgcgattttcgccgcctgcgcgaactgattgaa
agcggcgatggcgcggcgcagctggcgtatagcgtgtttacccatcgcctgcgcaaatat attggcgcgtatctggcggtgctgggccataccgatgtgattagctttaccgcgggcatt
ggcgaaaacgatgcggcggtgcgccgcgatgcggtgagcggcatggaagaactgggcatt gtgctggatgaacgccgcaacctggcgggcggcaaaggcgcgcgccagattagcgcggat
gatagcccgattaccgtgctggtggtgccgaccaacgaagaactggcgattgcgcgcgat tgcgtgcgcgtgctgggcggc
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):